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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique
Sei Saitoh1, Nobuo Terada, Nobuhiko Ohno
1Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
Abstract:
Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an "in vivo cryotechnique" (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofixed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffinized sections were immunostained for P-PK-S, Na(+)/HCO(3) (-) cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identified the same 40-kDa bands. The IVCT is useful for time-dependent analysis of the immunodistribution of P-PK-S and P-Akt-S.
Insights
The in vivo cryotechnique (IVCT) effectively captures real-time changes in protein kinase signaling within mouse kidneys during hypoxia. This method reveals dynamic shifts in phospho-PKA/C substrate and phospho-Akt substrate localization.
Area of Science:
- Renal Physiology
- Cell Signaling
- Biochemistry
Background:
- Protein kinases (PKs) are crucial for signal transduction via protein phosphorylation.
- Understanding the temporal regulation of PKs in vivo is essential for studying cellular responses to stimuli like hypoxia.
Purpose of the Study:
- To investigate the time-dependent regulation and in vivo immunolocalization of phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) in mouse kidneys under hypoxic conditions.
- To evaluate the utility of the "in vivo cryotechnique" (IVCT) for capturing these dynamic changes.
Main Methods:
- Normal and hypoxic mouse kidneys were prepared using the "in vivo cryotechnique" (IVCT).
- Kidneys were cryofixed, freeze-substituted, and examined using immunohistochemistry for P-PK-S, Na(+)/HCO(3) (-) cotransporter NBC1, and protein 4.1B.
- Immunoblotting was performed on kidney tissues under normal and hypoxic conditions.
Main Results:
- In normal kidneys, P-PK-S was cytoplasmic, while NBC1 and 4.1B were at basal striations of proximal tubules.
- Hypoxia induced a time-dependent shift in P-PK-S localization from cytoplasm to basal striations within 1-2 minutes.
- P-Akt-S exhibited similar localization patterns to P-PK-S under both normal and hypoxic conditions.
- Immunoblotting confirmed consistent 40-kDa bands under varying oxygen levels.
Conclusions:
- The IVCT is a valuable tool for the time-dependent analysis of protein kinase immunodistribution in vivo.
- Hypoxia triggers rapid, dynamic changes in the localization of P-PK-S and P-Akt-S in renal proximal tubules.
- These findings provide insights into the rapid signaling responses of kidney cells to oxygen deprivation.
